The present invention relates to therapeutic methods for the treatment and prevention of diseases via stimulation of a host""s immune system. Specifically, the invention relates to methods comprising the use of cationic amphipathic peptides having an xcex1-helical structure and which effect activation of cells of the monocyte/macrophage lineage and/or other lymphoid cells in a human or a non-human animal. The methods and compositions of the present invention are useful for the treatment and prevention of a variety of diseases, including, but not limited to, infectious diseases and cancer.
In the last few years, a large number of peptides have been identified sharing the characteristic of having an antimicrobial activity. One particular class comprises cationic amphipathic peptides which tend to have an xcex1-helical structure, especially in a low-polarity environment.
It is well-established that such antimicrobial peptides function through a lytic/ionophoric mechanism. Lehrer et al., 1993, Ann. Rev. Immunol. 11:105-128; Christensen et al., 1988, Proc. Natl. Acad. Sci. USA 85:5072-5076; Cruciani et al., 1991, Proc. Natl. Acad. Sci. USA 88:3792-3796; Viljanen et al., 1988, Infect. Immun. 56:3724-3730; Skerlavaj et al., 1990, Infect. Immun. 58:3724-3730; Okada and Natori, 1985, Biochem J. 229:453-458; Matsuyama and Natori, 1990, J. Biochem. 108:128-132; Keppi et al., 1989, Arch. Insect. Biochem. Physiol. 10:229-239; Ohta et al., 1992, Agents Chemother. 36:1460-1465. A common theme among these xe2x80x9clyticxe2x80x9d peptides is their permeabilizing effect by xe2x80x9cpunching holesxe2x80x9d into bacterial cytoplasmic membranes. The cationic, amphipathic structure of these peptides appears to facilitate the formation of hydrophilic ion channels in a lipid bilayer whereby the polar amino acids are positioned on one surface of the helix, and the apolar amino acids are positioned on the opposite side of the helix. Lee et al., 1986, Biochim. Biophys. Acta 862:211-219.
One family of these peptides with known antimicrobial properties, namely the dermaseptins, has been isolated from the skin of the tree frog, Phyllomedusa sauvagei. Others of this family have been isolated in later stages from Ph. bicolor. Mor et al., 1991, Biochemistry 30:8824; Mor et al., 1994, Biochemistry 33:6642; Mor et al., 1994, Eur. J. Biochem. 219:145. Each member of this family is a cationic amphipathic peptide with an xcex1-helical structure and is endowed with lytic activity against a wide array of pathogenic microorganisms in vitro.
Another peptide family with similar properties comes from well known, ubiquitous neuropeptides with functional and structural characteristics similar to the dermaseptins. Neuropeptide Y (NPY) (Tatemoto et al., 1982, Nature 296:659-660) and peptide YY (PYY) (Tatemoto, 1982, Proc. Natl. Acad. Sci. USA 79:2514-24518) two 36-residue peptides, are members of the pancreatic peptide (PP) family, found in the brain and in the lining of the gastrointestinal tract, respectively. They are involved in a variety of important regulatory functions and possess common features of tertiary structure, the so-called PP-fold. Glover et al., 1985, Eur. J. Biochem. 142:379-3385. The PP-fold, as characterized by X-ray diffraction analysis of crystals, consists of two antiparallel helices: an N-terminal polyproline helix and a long amphipathic xcex1-helix. To date, all PP family members were reported to induce their various biological effects by activating specific membrane bound receptors. Wahlestedt and Reis, 1993, Annu. Rev. Pharmacol. Toxicol. 32:309-352.
Other cationic amphipathic peptides having antimicrobial activity can be found, for example, among many other places, in the PCT Applications WO94/19369, published Sep. 1, 1994; U.S. Pat. No. 5,348,942.
Notably, the Minimal Inhibitory Concentration (MIC) for such peptides in order to exhibit lyticlionophoric, antimicrobial activity has been reported to be in the micromolar range. For example, as it is specifically reported in the U.S. Pat. No. 5,221,664, the MIC value for the antimicrobial peptide xe2x80x9cB13-33xe2x80x9d in order to exhibit antibacterial activity against Staphylococcus aureus is at least four (4) micromolar, the MIC for xe2x80x9cMagainin IIxe2x80x9d against Pseudonomas aeruginosa is as high as 256 micromolar. The effective concentrations can only be lowered by the addition of synergistic acting toxic cations, for example silver nitrate (see, U.S. Pat. No. 5,221,664).
Many infectious agents such as E. coli and S. aureus are pathogenic by virtue of their ability to proliferate in the circulation and in tissue space. These pathogens do not invade host organism cells and hence do not replicate as intracellular agents. As such, these types of pathogens are amenable to eradication by antibiotics, including peptide antimicrobials, that have no ability to enter mammalian cells. In contrast, certain pathogens such as M. tuberculosis, M. avium and M. intracellulare, and Leishmania sp. propagate primarily inside host organism cells, and in particular circulating cells of the immune system such as macrophages. These organisms are not accessible to the direct lytic effects of antimicrobial agents such as antimicrobial peptides that do not readily penetrate the infected mammalian cell.
The present invention is concerned with a novel use of cationic amphipathic peptides for therapeutic methods. As will be described hereinbelow, such peptides are useful for new methods for stimulating a host""s immune system by effecting the activation of cells of the monocyte/macrophage lineage and/or other lymphoid cells. These activated cells then contribute to the elimination of the pathogen. The amount of such peptides required for the methods of the present invention is significantly lower compared to the amount necessary for the lysis of bacterial cells.
The present invention is directed to methods for treating and/or preventing diseases said method comprising the administration to a host an active peptide having a stimulatory effect on the host""s immune system. Specifically, the cationic, amphipathic xcex1-helical peptides useful in the invention are pharmaceutically active by effecting activation of cells of the monocyte/macrophage lineage and/or other lymphoid cells in a treated human or non-human animal. Preferably, the peptides used have a length of about eight (8) to about fifty (50) amino acid residues.
In one embodiment, the peptide of the invention may have one of the following sequences:
(X)a(Z)n(X)b
and pharmaceutically acceptable salts thereof, wherein:
Z is selected from the primary sequences A-B-C-D, D-A-B-C, D-C-B-A, C-B-A-D, whereby each Z group within one peptide may be identical or different, and whereby Zn is positively charged and contains about 20% to about 50% hydrophilic amino acid residues, preferably about 25% to about 45% hydrophilic amino acid residues;
A is a hydrophobic or a small amino acid residue, whereby at least one A of two adjacent Z groups is hydrophobic;
C is a hydrophilic or a small amino acid residue, preferably a basic or neutral hydrophilic amino acid residue, whereby at least one C of two adjacent Z groups is basic hydrophilic;
B and D can be any amino acid residue, whereby B and D may be the same or different;
A, B, C, and D of each group may be the same or may be different in some or all of the groups;
(X)a and (X)b are amino acid assemblies of any length and composition which may not significantly contribute to the xcex1-helical structure;
nxe2x89xa72 and a,bxe2x89xa70, with the proviso that 8xe2x89xa6a+b+4nxe2x89xa650.
In another embodiment, the peptide of the invention may have one of the following sequences:
(X)a[(Z)n(X)c]d(Z)m(X)b
and pharmaceutically acceptable salts thereof,
wherein:
Z is selected from the primary sequences A-B-C-D, D-A-B-C, D-C-B-A, C-B-A-D, whereby each Z group within one peptide may be identical or different, and whereby Zn and Zm are positively charged and contain about 20% to about 50% hydrophilic amino acid residues, preferably about 25% to about 45% hydrophilic amino acid residues;
A is a hydrophobic or a small amino acid residue, whereby at least one A of two adjacent Z groups is hydrophobic;
C is a hydrophilic or a small amino acid residue, preferably a basic or neutral hydrophilic amino acid residue, whereby at least one C of two adjacent Z groups is basic hydrophilic;
B and D can be any amino acid residue, whereby B and D may be the same or different;
A, B, C, and D of each group may be the same or may be different in some or all of the groups;
(X)a, (X)b and (X)c are amino acid assemblies of any length and composition which may not significantly contribute to the xcex1-helical structure;
n,m,dxe2x89xa71 and a,b,cxe2x89xa70, with the proviso that 8xe2x89xa6a+b+d(c+4n)+4mxe2x89xa650.
According to the present invention, the peptides are administered to a host in an amount effective to activate cells of the monocytes/macrophage lineage and/or other lymphoid cells of said host. Preferably, peptides of the invention are administered in an amount effective to achieve a serum peptide level of about 10xe2x88x929 M to about 10xe2x88x925 M, typically the amount administered will be to achieve a serum peptide level of about 10xe2x88x929 M to about 10xe2x88x926 M. Such serum levels may be achieved by the administration of about 0.0005 to about 5.0 mg/kg body weight, typically about 0.0005 to about 0.5 mg/kg body weight. In some embodiments, the peptides are administered in combination with other compounds, including, but not limited to, antibiotics or protease inhibitors. The methods of the present invention are useful for the treatment of a variety of diseases including, but not limited to, infectious diseases and cancer.